Method of Treating Peripheral Nerve Disorders

ABSTRACT

The invention relates to the use of agents that bind the complement protein C5 in the treatment of diseases associated with inappropriate complement activation, and in particular in the treatment of peripheral nerve disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a division of U.S. patent application Ser.No. 12/440,462, a National Stage Entry of International PatentApplication No. PCT/GB2007/003401, filed on Sep. 10, 2007, which claimspriority to GB 0617734.9, filed on Sep. 8, 2006 (now abandoned), theentire contents of each of which are hereby incorporated by referenceherein.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named“2011203-0022_Sequence_Listing” on Nov. 12, 2015). The .txt file wasgenerated on Nov. 12, 2015 and is 2.6 kilobytes in size. The entirecontents of the Sequence Listing are herein incorporated by reference.

The present invention relates to the use of agents that bind thecomplement protein C5 in the treatment of diseases associated withinappropriate complement activation, and in particular in the treatmentof peripheral nerve disorders.

All documents mentioned in the text and listed at the end of thisdescription are incorporated herein by reference.

BACKGROUND TO THE INVENTION

The complement system is an essential part of the body's natural defencemechanism against foreign invasion and is also involved in theinflammatory process. More than 30 proteins in serum and at the cellsurface are involved in complement system function and regulation.Recently it has become apparent that, as well as the ˜35 knowncomponents of the complement system which may be associated with bothbeneficial and pathological processes, the complement system itselfinteracts with at least 85 biological pathways with functions as diverseas angiogenesis, platelet activation, glucose metabolism andspermatogenesis [1].

The complement system is activated by the presence of foreign antigens.Three activation pathways exist: (1) the classical pathway which isactivated by IgM and IgG complexes or by recognition of carbohydrates;(2) the alternative pathway which is activated by non-self surfaces(lacking specific regulatory molecules) and by bacterial endotoxins; and(3) the lectin pathway which is activated by binding of manna-bindinglectin (MBL) to mannose residues on the surface of a pathogen. The threepathways comprise parallel cascades of events that result in theproduction of complement activation through the formation of similar C3and C5 convertases on cell surfaces resulting in the release of acutemediators of inflammation (C3a and C5a) and formation of the membraneattack complex (MAC). The parallel cascades involved in the classicaland alternative pathways are shown in FIG. 1.

Complement can be activated inappropriately under certain circumstancesleading to undesirable local tissue destruction. Inappropriatecomplement activation has been shown to play a role in a wide variety ofdiseases and disorders including acute pancreatitis, Alzheimer'sdisease, allergic encephalomyelitis, allotransplatation, asthma, adultrespiratory distress syndrome, burn injuries, Crohn's disease,glomerulonephritis, haemolytic anaemia, haemodialysis, hereditaryangioedema, ischaemia reperfusion injuries, multiple system organfailure, multiple sclerosis, myasthenia gravis, ischemic stroke,myocardial infarction, psoriasis, rheumatoid arthritis, septic shock,systemic lupus erythematosus, stroke, vascular leak syndrome,transplantation rejection and inappropriate immune response incardiopulmonary bypass operations. Inappropriate activation of thecomplement system has thus been a target for therapeutic interventionfor many years and numerous complement inhibitors targeting differentparts of the complement cascade are under development for therapeuticuse.

In ischemic stroke and myocardial infarction, the body recognises thedead tissue in the brain or heart as foreign and activates complement socausing further local damage. Similarly in cardiopulmonary bypassoperations, the body recognises the plastic surfaces in the machine asforeign, activates complement and can result in vascular damage. Inautoimmune diseases, the body may wrongly recognise itself as foreignand activate complement with local tissue damage (e.g. joint destructionin rheumatoid arthritis and muscle weakness in myasthenia gravis).

In the peripheral nervous system, several types of neuropathy areautoimmune in origin and circulating autoantibodies to myelin andSchwann cell antigens have been detected [2]. Complement is implicatedas an effector in inflammatory demyelination observed in experimentalallergic neuritis (EAN), a model for Guillain-Barre syndrome, animmune-mediated acquired human demyelinating neuropathy [3].

Peripheral nerve disorders can be chronic and come on very slowly overseveral months or years. An example of such an illness is chronicinflammatory demyelinating polyradiculoneuropathy, known as CIDP.Sometimes peripheral nerve disorders are acute and come on very rapidlyover the course of a few days, for example post-infective demyelinatingpolyradiculoneuropathy, better known as Guillain-Barre Syndrome (GBS).CIDP was once known as ‘chronic GBS’ and is regarded as a relatedcondition to GBS.

GBS is a rare condition (prevalence is 1-2 per 100,000 or ˜3,000 casesin the USA per year). About half the cases of GBS occur after abacterial or viral infection. GBS is an autoimmune disorder in which thebody produces antibodies that damage the myelin sheath that surroundsperipheral nerves. The myelin sheath is a fatty substance that surroundsaxons, which increases the speed at which signals travel along thenerves.

Once these antibodies react with the antigen (the myelin sheath) thecomplement system is activated and C5 is produced. C5 breaks down to C5aand C5b which becomes C5b-9 (the membrane attack complex). C5a attractswhite cells and C5b-9 opens blood vessels wall to allow white cellingress into the tissues. Suitably stimulated white cells releasedestructive cytokines causing local damage to the nerves.

Clinically, GBS is characterised by weakness and numbness or tingling inthe legs and arms, and possible loss of movement and feeling in thelegs, arms, upper body, and face. The first symptoms of GBS are usuallynumbness or tingling (paresthesia) in the toes and fingers, withprogressive weakness in the arms and legs over the next few days. Somepatients experience paresthesia only in their toes and legs; others onlyexperience symptoms on one side of the body.

The symptoms may stay in this phase, causing only mild difficulty inwalking, requiring crutches or a walking stick. However, sometimes theillness progresses, leading to complete paralysis of the arms and legs.About one quarter of the time, the paralysis continues up the chest andfreezes the breathing muscles, leaving the patient dependant on aventilator. If the swallowing muscles are also affected, a feeding tubemay be needed.

GBS is considered a medical emergency and most patients are admitted tointensive care soon after diagnosis. Though GBS can improvespontaneously, there are a number of treatments that facilitaterecovery. Most patients with GBS and CIDP are treated withplasmapheresis (blood plasma exchange) or large doses of immunoglobulin.In extreme cases filtration of the CerebroSpinal Fluid has been used.Ventilation, plasmapheresis and immunoglobulin are extremely expensive.

There is thus a great need for agents that improve upon the currentlyavailable treatments for peripheral nerve disorders such as CIDP andGBS.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a method of treating or preventing aperipheral nerve disorder comprising administering to a subject in needthereof a therapeutically or prophylactically effective amount of anagent that binds complement C5.

The invention also provides the use of a therapeutically orprophylactically effective amount of an agent that binds complement C5in the manufacture of a medicament for treating or a preventingperipheral nerve disorder.

The peripheral nerve disorder of the present invention is selected fromthe group consisting of post-infective demyelinatingpolyradiculoneuropathy (Guillain Barre syndrome), Miller Fishersyndrome, acute inflammatory demyelinating polyradiculoneuropathy(AIDP), chronic inflammatory demyelinating polyradiculoneuropathy(CIDP), diabetic neuropathy, uraemic pruritus, multifocal motorneuropathy, paraproteinaemic neuropathy, anti-Hu neuropathy,post-diphtheria demyelinating neuropathy, multiple sclerosis, radiationmyelopathy, giant cell arteritis (temporal arteritis), transversemyelitis, motor neurone disease, dermatomyositis.

Preferably, the peripheral nerve disorder is selected from the groupconsisting of Guillain Barré Syndrome, chronic inflammatorydemyelinating polyradiculoneuropathy.

Preferably, the agent acts to prevent the cleavage of complement C5 byC5 convertase into complement C5a and complement C5b-9.

The complement C5 protein, also referred to herein as C5, is cleaved bythe C5 convertase enzyme, itself formed from C3a, an earlier product ofthe alternative pathway (FIG. 1). The products of this cleavage includean anaphylatoxin C5a and a lytic complex C5b-9 also known as membraneattack complex (MAC). C5a is a highly reactive peptide implicated inmany pathological inflammatory processes including neutrophil andeosinophil chemotaxis, neutrophil activation, increased capillarypermeability and inhibition of neutrophil apoptosis [4].

MAC is associated with other important pathological processes includingrheumatoid arthritis [5; 6], proliferative glomerulonephritis [7],idiopathic membranous nephropathy [8], proteinurea [9], demyelinationafter acute axonal injury [10] and is also responsible for acute graftrejection following xenotransplantation [11].

C5a has become a target of particular interest in the field ofcomplement-associated disorders [12]. Although C5a has manywell-recognised pathological associations, the effects of its depletionin humans appear to be limited. Monoclonal antibodies and smallmolecules that bind and inhibit C5a or C5a receptors have been developedto treat various autoimmune diseases. These molecules do not, however,prevent the release of MAC.

In contrast, administration of an agent that binds C5 according to thefirst aspect of the invention, inhibits both the formation of C5apeptide and the MAC. Surprisingly, it has been found that inhibition ofboth C5a and the MAC reduces the clinical symptoms associated withperipheral nerve disorders. Furthermore, because C5 is a late product ofthe classical and alternative complement pathways, inhibition of C5 isless likely to be associated with risks of concomitant infection thatexist when targeting earlier products in the cascade [13].

The ability of an agent to bind C5 may be determined by standard invitro assays known in the art, for example by western blotting followingincubation of the protein on the gel with labelled C5. Preferably, theagent according to the invention binds C5 with an IC₅₀ of less than 0.2mg/ml, preferably less than 0.1 mg/ml, preferably less than 0.05 mg/ml,preferably less than 0.04 mg/ml, preferably less than 0.03 mg/ml,preferably 0.02 mg/ml, preferably less than 1 μg/ml, preferably lessthan 100 ng/ml, preferably less than 10 ng/ml, more preferably still,less than 1 ng/ml.

Preferably, the agent that binds C5 is derived from a haematophagousarthropod. The term “haematophagous arthropod” includes all arthropodsthat take a blood meal from a suitable host, such as insects, ticks,lice, fleas and mites. Preferably, the agent is derived from a tick,preferably from the tick Ornithodoros moubata.

According to one embodiment of the invention, the agent that binds C5 isa protein comprising amino acids 19 to 168 of the amino acid sequence inFIG. 2 or is a functional equivalent of this protein. The agent thatbinds C5 may be a protein consisting of amino acids 19 to 168 of theamino acid sequence in FIG. 2 or be a functional equivalent of thisprotein.

According to an alternative embodiment, the protein used according tothis embodiment of the invention may comprise or consist of amino acids1 to 168 of the amino acid sequence in FIG. 2, or be a functionalequivalent thereof. The first 18 amino acids of the protein sequencegiven in FIG. 2 form a signal sequence which is not required for C5binding activity and so this may optionally be dispensed with, forexample, for efficiency of recombinant protein production.

The protein having the amino acid sequence given in FIG. 2, alsoreferred to herein as the EV576 protein, was isolated from the salivaryglands of the tick Ornithodoros moubata. EV576 is an outlying member ofthe lipocalin family and is the first lipocalin family member shown toinhibit complement activation. The EV576 protein inhibits thealternative, classical and lectin complement pathways by binding C5 andpreventing its cleavage by C5 convertase into Complement C5a andComplement C5b-9, thus inhibiting both the action of C5a peptide and theMAC. The term “EV576 protein”, as used herein, refers to the sequencegiven in FIG. 2 with or without the signal sequence.

The EV576 protein and the ability of this protein to inhibit complementactivation has been disclosed in [19], where the EV576 protein wasreferred to as the “OmCI protein”. It has now been found that the EV576protein is surprisingly effective in the treatment and prevention ofperipheral nerve disorders. The data presented herein demonstrate thatEV576 reduces the degree of clinical disease even when given during theactive disease phase in experimental autoimmune neuritis (EAN) in rats.EV576 thus represents a potential human therapy for the treatment andprevention of peripheral nerve disorders.

The surprising effectiveness of EV576 in the treatment of peripheralnerve disorders appears to be due to the fact that it acts by bindingC5, thus inhibiting the formation of C5a and MAC.

According to a further embodiment of the invention, the agent may be anucleic acid molecule encoding the EV576 protein or a functionalequivalent thereof. For example, gene therapy may be employed to effectthe endogenous production of the EV576 protein by the relevant cells inthe subject, either in vivo or ex vivo. Another approach is theadministration of “naked DNA” in which the therapeutic gene is directlyinjected into the bloodstream or into muscle tissue.

Preferably, such a nucleic acid molecule comprises or consists of bases53 to 507 of the nucleotide sequence in FIG. 2. This nucleotide sequenceencodes the EV576 protein in FIG. 2 without the signal sequence. Thefirst 54 bases of the nucleotide sequence in FIG. 2 encode the signalsequence of which is not required for complement inhibitory activity.Alternatively, the nucleic acid molecule may comprise or consist ofbases 1 to 507 of the nucleic acid sequence in FIG. 2, which encodes theprotein with the signal sequence.

The EV576 protein has been demonstrated to bind to C5 and prevent itscleavage by C5 convertase in rat, mouse and human serum with an IC₅₀ ofapproximately 0.02 mg/ml. Preferably, functional equivalents of theEV576 protein which retain the ability to bind C5 with an IC₅₀ of lessthan 0.2 mg/ml, preferably less than 0.1 mg/ml, preferably less than0.05 mg/ml, preferably less than 0.02 mg/ml, preferably less than 1μg/ml, preferably less than 100 ng/ml, preferably less than 10 ng/ml,more preferably still, less than 1 ng/ml.

In one respect, the term “functional equivalent” is used herein todescribe homologues and fragments of the EV576 protein which retain itsability to bind C5, and to prevent the cleavage of complement C5 by C5convertase into complement C5a and complement C5b-9. The term“functional equivalent” also refers to molecules that are structurallysimilar to the EV576 protein or that contain similar or identicaltertiary structure, particularly in the environment of the active siteor active sites of the EV576 protein that binds to C5, such as syntheticmolecules.

The term “homologue” is meant to include reference to paralogues andorthologues of the EV576 sequence that is explicitly identified in FIG.2, including, for example, the EV576 protein sequence from other tickspecies, including Rhipicephalus appendiculatus, R. sanguineus, R.bursa, A. americanum, A. cajennense, A. hebraeum, Boophilus microplus,B. annulatus, B. decoloratus, Dermacentor reticulatus, D. andersoni, D.marginatus, D. variabilis, Haemaphysalis inermis, Ha. leachii, Ha.punctata, Hyalomma anatolicum anatolicum, Hy. dromedarii, Hy. marginatummarginatum, Ixodes ricinus, I. persulcatus, I. scapularis, I. hexagonus,Argas persicus, A. reflexus, Ornithodoros erraticus, O. moubata moubata,O. m. porcinus, and O. savignyi. The term “homologue” is also meant toinclude the equivalent EV576 protein sequence from mosquito species,including those of the Culex, Anopheles and Aedes genera, particularlyCulex quinquefasciatus, Aedes aegypti and Anopheles gambiae; fleaspecies, such as Ctenocephalides felis (the cat flea); horseflies;sandflies; blackflies; tsetse flies; lice; mites; leeches; andflatworms. The native EV576 protein is thought to exist in O. moubata inanother three forms of around 18 kDa and the term “homologue” is meantto include these alternative forms of EV576.

Methods for the identification of homologues of the EV576 sequence givenin FIG. 2 will be clear to those of skill in the art. For example,homologues may be identified by homology searching of sequencedatabases, both public and private. Conveniently, publicly availabledatabases may be used, although private or commercially-availabledatabases will be equally useful, particularly if they contain data notrepresented in the public databases. Primary databases are the sites ofprimary nucleotide or amino acid sequence data deposit and may bepublicly or commercially available. Examples of publicly-availableprimary databases include the GenBank database(http://www.ncbi.nlm.nih.gov/), the EMBL database(http://www.ebi.ac.uk/), the DDBJ database (http://www.ddbj.nig.ac.jp/),the SWISS-PROT protein database (http://expasy.hcuge.ch/), PIR(http://pir.georgetown.edu/), TrEMBL (http://www.ebi.ac.uk/), the TIGRdatabases (see http://www.tigr.org/tdb/index.html), the NRL-3D database(http://www.nbrfa.georgetown.edu), the Protein Data Base(http://www.rcsb.org/pdb), the NRDB database(ftp://ncbi.nlm.nih.gov/pub/nrdb/README), the OWL database(http://www.biochem.ucl.ac.uk/bsm/dbbrowser/OWL/) and the secondarydatabases PROSITE (http://expasy.hcuge.ch/sprot/prosite.html), PRINTS(http://iupab.leeds.ac.uk/bmb5dp/prints.html), Profiles(http://ulrec3.unil.ch/software/PFSCAN_form.html), Pfam(http://www.sanger.ac.uk/software/pfam), Identify(http://dna.stanford.edu/identify/) and Blocks(http://www.blocks.fhcrc.org) databases. Examples ofcommercially-available databases or private databases includePathoGenome (Genome Therapeutics Inc.) and PathoSeq (IncytePharmaceuticals Inc.).

Typically, greater than 30% identity between two polypeptides(preferably, over a specified region such as the active site) isconsidered to be an indication of functional equivalence and thus anindication that two proteins are homologous. Preferably, proteins thatare homologues have a degree of sequence identity with the EV576 proteinsequence identified in FIG. 2 of greater than 60%. More preferredhomologues have degrees of identity of greater than 70%, 80%, 90%, 95%,98% or 99%, respectively with the EV576 protein sequence given in FIG.2. Percentage identity, as referred to herein, is as determined usingBLAST version 2.1.3 using the default parameters specified by the NCBI(the National Center for Biotechnology Information;http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=11 andgap extension penalty=1].

Homologues of the EV576 protein sequence given in FIG. 2 include mutantscontaining amino acid substitutions, insertions or deletions from thewild type sequence, for example, of 1, 2, 3, 4, 5, 7, 10 or more aminoacids, provided that such mutants retain the ability to bind C5. Mutantsthus include proteins containing conservative amino acid substitutionsthat do not affect the function or activity of the protein in an adversemanner. This term is also intended to include natural biologicalvariants (e.g. allelic variants or geographical variations within thespecies from which the EV576 proteins are derived). Mutants withimproved ability to bind C5 may also be designed through the systematicor directed mutation of specific residues in the protein sequence.

Fragments of the EV576 protein and of homologues of the EV576 proteinare also embraced by the term “functional equivalents” providing thatsuch fragments retain the ability to bind C5. Fragments may include, forexample, polypeptides derived from the EV576 protein sequence which areless than 150 amino acids, less than 125 amino acids, less than 100amino acids, less than 75 amino acids, less than 50 amino acids, or even25 amino acids or less, provided that these fragments retain the abilityto bind to complement C5. Included as such fragments are not onlyfragments of the O. moubata EV576 protein that is explicitly identifiedherein in FIG. 2, but also fragments of homologues of this protein, asdescribed above. Such fragments of homologues will typically possessgreater than 60% identity with fragments of the EV576 protein sequencein FIG. 2, although more preferred fragments of homologues will displaydegrees of identity of greater than 70%, 80%, 90%, 95%, 98% or 99%,respectively with fragments of the EV576 protein sequence in FIG. 2.Fragments with improved may, of course, be rationally designed by thesystematic mutation or fragmentation of the wild type sequence followedby appropriate activity assays. Fragments may exhibit similar or greateraffinity for C5 as EV576 and may have the same or greater IC₅₀ for C5.

A functional equivalent used according to the invention may be a fusionprotein, obtained, for example, by cloning a polynucleotide encoding theEV576 protein in frame to the coding sequences for a heterologousprotein sequence. The term “heterologous”, when used herein, is intendedto designate any polypeptide other than the EV576 protein or itsfunctional equivalent. Example of heterologous sequences, that can becomprised in the soluble fusion proteins either at N- or at C-terminus,are the following: extracellular domains of membrane-bound protein,immunoglobulin constant regions (Fc region), multimerization domains,domains of extracellular proteins, signal sequences, export sequences,or sequences allowing purification by affinity chromatography. Many ofthese heterologous sequences are commercially available in expressionplasmids since these sequences are commonly included in the fusionproteins in order to provide additional properties without significantlyimpairing the specific biological activity of the protein fused to them[14]. Examples of such additional properties are a longer lastinghalf-life in body fluids, the extracellular localization, or an easierpurification procedure as allowed by a tag such as a histidine or HAtag.

The EV576 protein and functional equivalents thereof, may be prepared inrecombinant form by expression in a host cell. Such expression methodsare well known to those of skill in the art and are described in detailby [15] and [16]. Recombinant forms of the EV576 protein and functionalequivalents thereof are preferably unglycosylated.

The proteins and fragments of the present invention can also be preparedusing conventional techniques of protein chemistry. For example, proteinfragments may be prepared by chemical synthesis. Methods for thegeneration of fusion proteins are standard in the art and will be knownto the skilled reader. For example, most general molecular biology,microbiology recombinant DNA technology and immunological techniques canbe found in [15] or [17].

The subject to which the agent that binds C5 is administered in themethod or use of the invention is preferably a mammal, preferably ahuman. The subject to which the agent that binds C5 is administered mayalso be suffering from a further disease with which peripheral nervedisorders are associated, such as diabetes mellitus, vasculitis,paraproteinaemia and hereditary motor and sensory neuropathy.

The agent is administered in a therapeutically or prophylacticallyeffective amount. The term “therapeutically effective amount” refers tothe amount of agent needed to treat or ameliorate a targeted disease.The term “prophylactically effective amount” used herein refers to theamount of agent needed to prevent a targeted disease.

Preferably, the dose of the agent is sufficient to bind as muchavailable C5 as possible in the subject, more preferably, all availableC5. Preferably, the dose of the agent supplied is at least twice themolar dose needed to bind all available C5 in the subject. The dose ofthe agent supplied may be 2.5 times, 3 times or 4 times the molar doseneeded to bind all available C5 in the subject. Preferably, the dose isfrom 0.0001 mg/kg (mass of drug compared to mass of patient) to 20mg/kg, preferably 0.001 mg/kg to 10 mg/kg and more preferably 0.1 mg/kgto 1 mg/kg.

The frequency with which the dose needs to be administered will dependon the half-life of the agent involved. Where the agent is the EV576protein or a functional equivalent thereof, the dose may be administeredas a continuous infusion, in bolus doses or on a daily basis, twicedaily basis, or every two, three, four days, five, six, seven, 10, 15 or20 days or more.

The exact dosage and the frequency of doses may also be dependent on thepatient's status at the time of administration. Factors that may betaken into consideration when determining dosage include the severity ofthe disease state in the patient, the general health of the patient, theage, weight, gender, diet, time and frequency of administration, drugcombinations, reaction sensitivities and the patient's tolerance orresponse to therapy. The precise amount can be determined by routineexperimentation, but may ultimately lie with the judgement of theclinician.

The agent will generally be administered as part of a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”, asused herein, includes genes, polypeptides, antibodies, liposomes,polysaccharides, polylactic acids, polyglycolic acids and inactive virusparticles or indeed any other agent provided that the carrier does notitself induce toxicity effects or cause the production of antibodiesthat are harmful to the individual receiving the pharmaceuticalcomposition. Pharmaceutically acceptable carriers may additionallycontain liquids such as water, saline, glycerol, ethanol or auxiliarysubstances such as wetting or emulsifying agents, pH bufferingsubstances and the like. The pharmaceutical carrier employed will thusvary depending on the route of administration. Carriers may enable thepharmaceutical compositions to be formulated into tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions to aidintake by the patient. A thorough discussion of pharmaceuticallyacceptable carriers is available in [18].

The agent may be delivered by any known route of administration. Theagent may be delivered by a parenteral route (e.g. by injection, eithersubcutaneously, intraperitoneally, intravenously or intramuscularly ordelivered to the interstitial space of a tissue). The compositions canalso be administered into a lesion. Other modes of administrationinclude oral and pulmonary administration, suppositories, andtransdermal or transcutaneous applications, needles, and hyposprays.

In one embodiment the agent is administered intravenously at a dose of13 mg/kg followed by a 12-hourly dose of 4 mg/kg intraperitoneally.

The agent that binds C5 may be administered alone or as part of atreatment regimen also involving the administration of other drugscurrently used in the treatment of patients with a peripheral nervedisorder. For example, the agent may be administered in combination withthe infusion of immunoglobulin or in combination with plasmapheresistreatment.

Combinations of drug treatments may have an additive or synergisticeffect on treatment of the disease.

The invention thus provides—(i) an agent that binds C5, preferably theEV576 protein or a functional equivalent thereof, and (ii)immunoglobulin, for use in therapy.

The invention also provides the use of—(i) an agent that binds C5,preferably the EV576 protein or a functional equivalent thereof, and(ii) immunoglobulin, in the manufacture of a medicament for treating aperipheral nerve disorder.

The agent that binds C5 may be administered simultaneously, sequentiallyor separately with the other drug(s). For example, the agent that bindsC5 may be administered before or after administration of the otherdrug(s).

The invention thus provides the use of an agent that binds C5,preferably the EV576 protein or a functional equivalent thereof, in themanufacture of a medicament for treating a peripheral nerve disorder ina subject, wherein said subject has been pre-treated with animmunoglobulin. The invention also provides the use of an immunoglobulinin the manufacture of a medicament for treating a peripheral nervedisorder in a subject wherein said subject has been pre-treated with anagent that binds C5, preferably the EV576 protein or a functionalequivalent thereof.

The agent that binds C5 may also be administered as part of a treatmentregimen also involving the administration of other drugs currently usedin the treatment of other diseases with which a peripheral nervedisorder is associated, such as diabetes mellitus, vasculitis,paraproteinaemia and hereditary motor and sensory neuropathy. The agentthat binds C5 may be administered simultaneously, sequentially orseparately with the other drug(s). For example, the agent that binds C5may be administered before or after administration of the other drug(s).

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example. It will be appreciated thatmodification of detail may be made without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Schematic diagram of classical and alternative pathways ofcomplement activation. Enzymatic components, dark grey. Anaphylatoxinsenclosed in starbursts.

FIG. 2: Primary sequence of EV576. Signal sequence underlined. Cysteineresidues in bold type. Nucleotide and amino acid number indicated atright.

FIGS. 3A, 3B, 3C, and 3D: Purification of EV576 from tick salivary glandextract (SGE). FIG. 3A) Anion exchange chromatography. FIG. 3B)Classical haemolytic assay of fractions. FIG. 3C) Reducing SDS-PAGE.FIG. 3D) RP-HPLC.

FIGS. 4A, 4B, and 4C: Mechanism of action of EV576. FIG. 4A) No effecton C3a production. FIG. 4B) Prevents C5a production. FIG. 4C) Bindsdirectly to C5.

FIGS. 5A and 5B: Recombinant EV576. FIG. 5A) Recombinant EV576 (rEV576)inhibits complement as effectively as native EV576. FIG. 5B) Structureof EV576.

FIGS. 6A and 6B: Effect of rEV576 in experimental autoimmune neuritismodel. FIG. 6A) Weight loss in rEV576 treated animals compared withcontrol animals. FIG. 6B) Clinical scores in animals treated with rEV576compared with control animals.

EXAMPLES 1. Mechanism of Action and Inhibitory Concentration

EV576 was purified from salivary gland extracts of the soft tickOrthinodoros moubata by SDS-PAGE and RP-HPLC of fractions of salivarygland extract found to contain complement inhibitory activity byclassical haemolytic assays (FIG. 3) as disclosed in [19].

EV576 inhibits both human and guinea pig classical and alternativepathways. It has no effect on the rate of C3a production (FIG. 4A) butprevents cleavage of C5a from C5 (FIG. 4B).

The ability of EV576 to inhibit both the classical and the alternativecomplement pathways is due to binding of the molecule to complement C5,the precursor of C5a and C5b-9. EV576 binds directly to C5 (FIG. 4C)with an IC₅₀ of ≈0.02 mg/ml. The precise binding mechanism and accessoryroles (if any) played by serum factors are under investigation.

Recombinant EV576 (rEV576) with glycosylation sites removed (whichotherwise are glycosylated in the yeast expression system) is as activeas the native non-glycosylated protein (FIG. 5A).

The structure of EV576 confirms that it is an outlying member of thelipocalin family (FIG. 5B), having 46% identity with moubatin, aplatelet aggregation inhibitor from O. moubata. Lipocalins are a largegroup of soft tick proteins the functions of which, with rareexceptions, are unknown.

2. Effect of EV576 on Experimental Autoimmune Neuritis

Experimental autoimmune neuritis (EAN) was induced in rats according tothe method described in reference [20].

Lewis rats were injected with 170 μg peripheral nerve myelin P0 proteinpeptide 106-124 and 1.5 mg Mycobacterium tuberculosis with Freund'sincomplete adjuvant at day 0. At Day 11 after inoculation, 93% of theanimals had a score of 2 on the clinical scoring grade. The severity ofparesis (paralysis/weakness) was graded as follows: 0=no illness;1=flaccid tail; 2=moderate paraparesis; 3=severe paraparesis; and4=tetraparesis or death. At Day 11, the rats were injected with a) 3mg/rat intravenously followed by 1 mg/rat intraperitoneally at 12-hourintervals for 7 days, b) 0.3 mg/rat intravenously followed by 0.1 mg/ratintraperitoneally at 12-hour intervals for 7 days or c) PBS. Controlgroups were totally untreated. The treatment was limited to 7 days andthen stopped. The animals were then followed until day 38 when theanimals were euthanased. The rats were assessed for changes in weightand clinical grade.

All animals lost body weight over the initial 10-day period oftreatment. Weight was regained from Day 17-18 until Day 38 when theanimal's original body weight was regained (FIG. 6a ). There were nosignificant differences between treatment groups.

By Day 11, 93% of animals had a clinical score of 2 with a mean score of2.17. Treatment occurred from Day 11 to Day 18. On Day 17 and 18, bothactive treatment groups (i.e. high and low dose rEV576 treated groups,showed statistically significantly lower clinical scores than theuntreated group (P<0.001) and the PBS treated group (P<0.01). There wasno difference between the two active groups (that is, high and low doserEV576) (FIG. 6b ).

Thus rEV576 given by iv/ip injection reduces the degree of clinicaldisease even when given during the active disease phase (that is, at aclinical score of 2). Earlier treatment may show greater effect. ThusrEV576 represents a possible human therapy for peripheral nervedisorders such as GBS and CIDP.

-   [1] Mastellos, D., et al., Clin Immunol, 2005. 115(3): p. 225-35-   [2] Kwa., et. al., Brain 2003. 126, 361-375-   [3] Stoll, et. al., Ann Neurol 1991. 30:147-155-   [4] Guo, R. F. and P. A. Ward, Annu Rev Immunol, 2005. 23: p. 821-52-   [5] Neumann, E., et al., Arthritis Rheum, 2002. 46(4): p. 934-45-   [6] Williams, A. S., et al., Arthritis Rheum, 2004. 50(9): p.    3035-44-   [7] Quigg, R. J., Curr Dir Autoimmun, 2004. 7: p. 165-80-   [8] Papagianni, A. A., et al., Nephrol Dial Transplant, 2002.    17(1): p. 57-63-   [9] He, C., et al., J Immunol, 2005. 174(9): p. 5750-7-   [10] Mead, R. J., et al., J Immunol, 2002. 168(1): p. 458-65-   [11] Nakashima, S., et al., J Immunol, 2002. 169(8): p. 4620-7-   [12] Mizuno, M. and D. S. Cole, Expert Opin Investig Drugs, 2005.    14(7): p. 807-21-   [13] Allegretti, M., et al. Curr Med Chem, 2005. 12(2): p. 217-36-   [14] Terpe K, Appl Microbiol Biotechnol, 2003. 60: 523-33-   [15] Sambrook et al (2000)-   [16] Fernandez & Hoeffler (1998)-   [17] Ausubel et al. (1991)-   [18] Remington's Pharmaceutical Sciences; Mack Pub. Co., N. J. 1991-   [19] WO2004/106369-   [20] Zhu, J. et al. Acta Neurologica Scandinavica, 1994. 90: p.    19-25

1. A method of treating a peripheral nerve disorder comprisingadministering to a subject in need thereof a therapeutically effectiveamount of an agent that binds complement C5, wherein the agent is aprotein comprising or consisting of amino acids 19 to 168 of the aminoacid sequence in FIG.
 2. 2. Use of a therapeutically or prophylacticallyeffective amount of an agent that binds complement C5 in the manufactureof a medicament for treating or preventing a peripheral nerve disorder.3. The method according to claim 1 wherein the agent acts to prevent thecleavage of complement C5 by C5 convertase into complement C5a andcomplement C5b-9.
 4. The method according to claim 1 wherein the agentbinds C5 with an IC₅₀ of less than 0.2 mg/ml.
 5. The method according toclaim 1 wherein the agent is derived from a haematophagous arthropod. 6.(canceled)
 7. (canceled)
 8. A method of treating or preventing aperipheral nerve disorder comprising administering to a subject in needthereof a therapeutically or prophylactically effective amount of anucleic acid molecule encoding a protein comprising or consisting ofamino acids 19 to 168 of the amino acid sequence in FIG. 2 or afunctional equivalent thereof.
 9. The method according to claim 8wherein the nucleic acid molecule comprises or consists of bases 53 to507 of the nucleotide sequence in FIG.
 2. 10. The method according toclaim 9 wherein the nucleic acid molecule comprises or consists of bases1 to 507 of the nucleotide sequence in FIG.
 2. 11. The method accordingto claim 1 wherein the subject is a mammal, preferably a human.
 12. Themethod according to claim 1 wherein the agent is administered in a dosesufficient to bind as much available C5 as possible in the subject, morepreferably, all available C5.
 13. The method according to claim 1wherein the agent is administered intravenously at a dose of 13 mg/kgfollowed by a 12-hourly dose of 4 mg/kg intraperitoneally.
 14. Themethod according to claim 1 wherein the agent that binds C5 isadministered as part of a treatment regimen also involving theadministration of a further drug for the treatment of a peripheral nervedisorder.
 15. The method according to claim 14 wherein the further drugis immunoglobulin.
 16. The method according to claim 14 wherein theagent that binds C5 is administered simultaneously, sequentially orseparately with the further drug.
 17. A method according to claim 1wherein the peripheral nerve disorder is selected from the groupconsisting of post-infective demyelinating polyradiculoneuropathy(Guillain Barré syndrome), Miller Fisher syndrome, acute inflammatorydemyelinating polyradiculoneuropathy (AIDP), chronic inflammatorydemyelinating polyradiculoneuropathy (CIDP), diabetic neuropathy,uraemic pruritus, multifocal motor neuropathy, paraproteinaemicneuropathy, anti-Hu neuropathy, post-diphtheria demyelinatingneuropathy, multiple sclerosis, radiation myelopathy, giant cellarteritis (temporal arteritis), transverse myelitis, motor neuronedisease, dermatomyositis.
 18. A method according to claim 17 wherein theperipheral nerve disorder is selected from the group consisting ofGuillain Barré Syndrome, chronic inflammatory demyelinatingpolyradiculoneuropathy.
 19. A method of preventing a peripheral nervedisorder comprising administering to a subject in need thereof aprophylactically effective amount of a protein comprising or consistingof amino acids 19 to 168 of the amino acid sequence in FIG. 2.